Methods for diagnosis and treatment of chronic immune diseases

ABSTRACT

Methods are provided for diagnosing and/or characterizing chronic immune disease activity in a subject. In the subject methods, a sample is obtained from a subject suspected of having or known to have a chronic immune disease. The sample is then assayed for the presence of native Stat-1 protein and/or any lower molecular weight fragments of Stat-1 protein present. The assay results are used to diagnose the presence of chronic immune disease activity and/or characterize chronic immune disease activity in the subject, e.g., to confirm an initial chronic immune disease diagnosis, to determine the stage of the disease, to monitor disease progression, to predict disease attacks, and the like. In certain embodiments, the assay results are also used to predict the effectiveness of a particularly treatment protocol, e.g., to determine whether an interferon based treatment protocol will be effective. In addition, methods of Stat-1 based methods of treating chronic immune disease conditions are provided. Also provided by the subject invention are kits for practicing the methods.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to thefiling date of the U.S. Provisional Patent Application Ser. No.60/299,323 filed Jun. 19, 2001; the disclosure of which are hereinincorporated by reference.

INTRODUCTION

1. Technical Field

The field of invention is chronic immune disease, particularly multiplesclerosis and chronic fatigue syndrome.

2. Background of the Invention

Chronic immune diseases can be highly debilitating, often requiringtreatment. Two such chronic immune diseases are multiple sclerosis andchronic fatigue syndrome.

Multiple sclerosis (MS) is a neurological illness of unknown etiologyassociated with attacks of focal or multifocal neurological dysfunctionindicating lesions within the central nervous system (CNS). In Americaand Northern Europe, MS is the most common neurological disease, withprevalence rates estimated between 50-100 per 100,000 population. Theonset of disease is most common in early adulthood. Recurrent attackscan occur over many years, with approximately 30 percent of the patientsprogressing to a severe form of the disease which can be fatal.

MS is pleomorphic in its presentation. The clinical manifestations aredetermined in part by the location of the foci of demyelination withinthe CNS. Classical features of the disease include impaired vision,nystagmus, dysarthria, ataxia and intention tremor, andweakness/paralysis of one or more limbs. The demyelination is likely dueto an autoimmune, inflammatory response that results in the destructionof the myelin sheath covering the axon of the peripheral nerves in theCFS.

The most common form of the disease is episodic. Symptoms develop withsubsequent recovery, followed by another attack. In approximately 50percent of all patients with MS, attacks become more frequent, usuallywith a worsening of symptomatology. In 30 percent, the disease developsinto what is referred to as “progressive/relapsing,” the most severeform of the disease. In this state remissions are rare and patientsfrequently become wheelchair bound.

The diagnosis of MS remains problematic, and frequently the disease isnot diagnosed until the patient has experiences two or more “attacks.”To aid the clinician, the only laboratory test available is testing thecerebrospinal fluid for oligoclonal bands, present in approximately 90percent of all patients. Examination of the brain for demyelinatingplaques, using magnetic resonance imaging (MRI) is useful but expensiveand is not warranted except in a small group of patients in which allother clinical and laboratory tests are negative.

There is no diagnostic laboratory test to determine if a patient ishaving an “attack,” to monitor the progress of the “attack,” todetermine if the patient is progressing to a more active form of thedisease (i.e., progressive/relapsing), nor is any laboratory testavailable as a prognostic indicator and/or to monitor therapy ifadministered.

Chronic Fatigue Syndrome (CFS) is an illness of unknown etiology. CFS isoften associated with sudden onset, flu-like symptoms, debilitatingfatigue, low-grade fever, myalgia and neurocognitive dysfuntion. CFSpatients typically display reduced Karnofsky Performance (KPS) scores.The KPS measures an individual's ability to function and carry on normalactivities. KPS scores range from zero (0) for a completelynon-functional or dead patient to one hundred (100) for a completelynormal function.

Diagnosis of CFS remains one of exclusion. An accumulating body ofevidence suggests that CFS is associated with dysregulation of bothhumoral and cellular immunity, including mitogen response, reactivationof viruses, abnormal cytokine production, diminished natural killer cellfunction and changes in intermediary metabolites.

It has been suggested that the clinical and immunological abnormalitiesobserved in MS and CFS might be caused by defects in theinterferon-inducible pathways i.e., the 2′-5′-oligoadenylate (2-5A)synthetase/RNase L and p68 kinase (PKR) antiviral defense pathways(Suhadolnik et al., Clin. Infect. Dis. 18:S96-S104, 1994; Suhadolnik etal., In Vivo 8:599-604, 1994). The 2-5A synthetase/RNase L pathway ispart of the antiviral defense mechanism in mammalian cells (Lengyel,Ann. Rev. Biochem. 51:251-282, 1982; Sen et al., Adv. Virus Res.42:57-102, 1993).

When activated by dsRNA, 2-5A synthetase converts ATP to 2′-5′-linkedoligoadenylates with 5′ terminal phosphates. Biologically active 2-5Abinds to and activates a latent endoribonuclease, RNase L, which in turnhydrolyzes single-stranded cellular and viral RNA, primarily after UpNpsequences, thereby inhibiting protein synthesis. In addition,circulating white blood cells from patients with CFS have beendemonstrated to contain abnormal, low molecular weight forms of RNase L(Suhadolnik et al., J. Interferon & Cytokine Res. 17:377-385, 1997; DeMeirleir et al., Am. J. Med. 108:99-105, 2000).

The use of interferon (e.g., Roferon®, Betaseron®) or IFN-inducers(e.g., Ampligen®, Imiquimod®, etc.) as therapy for chronic immunediseases, in particular MS, has met with limited success. For interferonto induce its enzymatic pathways within the cells of the immune system,interferon must first bind to its receptor which in turn activates oneof the STAT (signal transducers and activators of transcription)proteins. Indeed STATs were discovered through the study of interferonsignaling. A unique feature of Stat-mediated signaling is that thepathway from cell membrane to nucleus is traversed by a single molecule,as opposed to a cascade of kinases and adaptor molecules (Hoey et al.,Current Opinion in Genetics and Development 8:582-587, 1998).

Stat-1 plays an important role in growth arrest, in promoting apoptosisand is implicated as a tumor suppressor (Bromberg et al., Oncogene19:2468-2473, 2000). Stat-1 null cells are resistant to apoptoticinduction by TNF-alpha, and re-introduction of Stat-1 restored bothTNF-alpha-induced apoptosis and the expression of caspases 1, 2, and 3.(Kumar et al., Science 278:1630-1632, 1997). A wide variety of virusesare known to encode proteins that specifically inhibit the activation ofthe IFN-mediated state and/or interact with specific IFN-inducedproteins causing a disruption of the IFN-mediated state (Alcami et al.,Trends in Microbiology 8:410-418, 2000).

Stat-1 deficient mice exhibit a severe defect in IFN-dependent immuneresponses against viruses and microbial pathogens. Treatment of normalmacrophages with IFN-alpha or IFN-gamma in the presence of LPS inducedthe secretion of nitric oxide (NO). In contrast, IFN-induced NOproduction was not observed in Stat-1 knockout macrophages. Thereforedefective macrophage activity led to a high sensitivity to viralinfections. (Durbin et al., Cell 84:443-450, 1996; Meraz et al., Cell84:431-442, 1996).

If Stat-1 is disabled or otherwise inactive in the cells of the immunesystem, treatment with interferon or interferon inducer will not beeffective in promoting and establishing the interferon-inducibleantiviral and antiproliferative pathways.

As the above discussion demonstrates, currently employed methods ofdiagnosing and/or characterizing MS or CFS disease activity in a subjectare inadequate. As such, there is a continued need in the field todevelop additional means for diagnosing and/or characterizing MS or CFSdisease activity in a subject. Of particular interest would be thedevelopment of methods that can be used to predict the effectiveness ofa given proposed treatment protocol. In addition, an effective cure foreither MS or CFS has yet to be developed. As such, there is continuedinterest in the identification of new treatment protocols for chronicimmune diseases, and particularly for MS and CFS, as well as thedevelopment of methods to make interferon based protocols moreeffective.

Relevant Literature

U.S. patents of interest include: U.S. Pat. Nos. 5,766,859; 5,776,690;5,830,668; 5,853,996, 5,985,565, 6,153,591, and 6,184,210. Also ofinterest is WO 91/00097. Other references of interest include: Komaroff,Am. J. Med. 108:69-71, 2000; Yokosawa et al., Arch. Virol.143:1985-1992, 1998; King et al., J. Biol. Chem. 273:8699-8704, 1998;Hoi-Tao et al., EBMO J. 16:1291-1304, 1997; Bottrel et al., Antimicrob.Agents. Chemother. 43:856-861, 1999; Yang et al., Proc. Natl. Acad. Sci.97:13631-13636, 2000.

SUMMARY OF THE INVENTION

Methods are provided for characterizing chronic immune disease activityin a subject. In the subject methods, a sample is obtained from asubject suspected of having or known to have a chronic immune disease.The sample is then assayed for the presence and amount of intact (i.e.,native) Stat-1 protein and/or fragments thereof. The assay results areused to diagnose the presence of chronic immune disease and/orcharacterize chronic immune disease activity in a subject. In addition,the assay results may used to evaluated the effectiveness of a giventreatment protocol, e.g., to determine whether or not to treat thesubject with interferon and/or inducers of interferon. Also provided aremethods of treating chronic immune disease via enhancement of Stat-1activity. Also provided by the subject invention are kits for practicingthe methods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents a densitometric scan of a Western blot detectingStat-1 protein and Stat-1 protein fragments from PBMC extracts from CFSpatients. The value indicated at the top of each lane is the ratio ofRNase L fragments as calculated by [(LMW/HMW)*10] as assayed in PBMCextracts from CFS patients.

FIG. 2 represents a densitometric scan of a Western blot detectingStat-1 protein from PBMC extracts of healthy controls and MS patients.

FIG. 3 represents a densitometric scan of a Western blot detecting Bcl-2as a control for the non-specific degradation of proteins in the PBMCextracts.

FIG. 4 represents a densitometric scan of a Western blot of PBMCextracts from healthy controls (i.e., RNase L ratio <0.2) mixed withPBMC extracts from CFS patients (i.e., RNase L ratio >2.0) in theabsence or presence of protease inhibitors.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods are provided for diagnosing and/or characterizing chronic immunedisease activity in a subject. In the subject methods, a sample isobtained from a subject suspected of having or known to have a chronicimmune disease. The sample is then assayed for the presence of nativeStat-1 protein and/or any lower molecular weight fragments of Stat-1protein present. The assay results are used to diagnose the presence ofchronic immune disease activity and/or characterize chronic immunedisease activity in the subject, e.g., to confirm an initial chronicimmune disease diagnosis, to determine the stage of the disease, tomonitor disease progression, to predict disease attacks, and the like.In certain embodiments, the assay results are also used to predict theeffectiveness of a particularly treatment protocol, e.g., to determinewhether an interferon based treatment protocol will be effective. Inaddition, methods of Stat-1 based methods of treating chronic immunedisease conditions are provided. Also provided by the subject inventionare kits for practicing the methods.

Before the invention is described further, it is to be understood thatthe invention is not limited to the particular embodiments of theinvention described below, as variations of the particular embodimentsmay be made and still fall within the scope of the appended claims. Itis also to be understood that the terminology employed is for thepurpose of describing particular embodiments, and is not intended to belimiting. Instead, the scope of the present invention will beestablished by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the invention componentswhich are described in the publications which might be used inconnection with the presently described invention.

As summarized above, the subject invention provides a method ofdiagnosing the presence of a chronic immune disease in a host. In otherwords, the subject invention provides a means for determining whether ahost is suffering from a chronic immune disease. Specifically, thesubject invention provides a method of determining whether a host issuffering from MS or CFS. MS and CFS are disease conditions known tothose of skill in the art, and are further defined above and below.

In determining whether a host suffers from a chronic immune disease, asample from the host is assayed for the presence of one or more lowmolecular weight fragments of Stat-1 protein (or Stat-1). By lowmolecular weight Stat-1 fragment is meant a polypeptide that has asequence of amino acid residues found in full length Stat-1, where thissequence is at least about 10, usually at least about 20 and moreusually at least about 50 residues long and is often longer, where thepolypeptide has a molecular weight that is less than that molecularweight of full length Stat-1, i.e., where the polypeptide has amolecular weight that is less than about 90 kDa, as measured by SDS-PAGE(see the Experimental Section, infra.) Specifically, the sample isassayed for low molecular weight Stat-1 fragments ranging in weight fromabout 15 to 75 kDa, usually from about 30 to 60 kDa and more usuallyfrom about 45 to 50 kDa. Of particular interest is the identification ofa Stat-1 fragment having a molecular weight of about 50 kDa asdetermined by SDS-PAGE. Representative samples and assay methods foridentifying the presence of, and amounts of, low molecular weight Stat-1fragments are described in greater detail infra.

The presence or absence of the low molecular weight Stat-1 fragments isthen used to diagnose whether or not the host suffers from the chronicimmune disease. In other words, the presence or absence of low molecularweight Stat-1 fragments in the sample is used to determine whether ornot the host suffers from a chronic immune disease, such as CFS or MS.For example, in one embodiment, the presence of one or more lowmolecular weight Stat-1 fragments is used to determine whether the hostsuffers from CFS. Likewise, in another embodiment, the presence of oneor more low molecular weight Stat-1 fragments is used to determinewhether a host suffers from MS. As part of the diagnosis, one may alsoevaluate the subject for other symptoms of the disease of interest whichis to be diagnosed, e.g. the MS or CFS symptoms described in thebackground section, supra, as well as in other parts of thisapplication.

Also provided by the subject invention are methods of characterizing thechronic immune disease activity, e.g. CFS or MS disease activity, in asubject suspected of having, or known to have, a chronic immune disease,e.g. CFS or MS. Subjects suspected of having, or known to have, achronic immune disease and thus amenable to the subject methods can beidentified using any convenient protocol. One convenient protocol isdiagnosis based on clinical symptoms. A number of different clinicalsymptoms may be used to identify subjects that may have or have thechronic immune disease of interest, where the specific symptoms employedwill necessarily depend on the specific chronic immune disease. Forexample, where the chronic immune disease of interest is CFS, clinicalsymptoms of interest include: fatigue of six months or longer thatcauses a reduction in effort of greater than 50 percent of normaloutput, athralgia, myalgia, sore throat accompanied by swollen glands,cognitive dysfunction (e.g. memory loss); and the like. For MS, clinicalsymptoms include: weakness of the limbs; sensory symptoms, e.g.paresthesia or hypesthesia; ataxia; optic neuritis; diplopia; trigeminalneuralgia; facial paralysis; vertigo; urinary or bowel movementabnormalities; and cognitive dysfunction, e.g. memory loss, impairedattention, problem-solving difficulties, slowed information processing,and difficulty in shifting between cognitive tasks. The presence of oneor more of the above symptoms may be used to identify subjects suspectedof suffering from CFS or MS, respectively. Other assays may also beemployed, including MRI imaging, the oligoclonal band assay described ingreater detail infra, etc.

The first step of the subject methods is to obtain a suitable samplefrom the subject or patient of interest, i.e. a patient suspected ofhaving or known to have the chronic immune disease of interest, e.g. CFSor MS. The sample is derived from any initial source that containsnative Stat-1 and the low molecular weight Stat-1 fragments (ifpresent). Sample sources of interest include, but are not limited to,many different physiological sources, e.g. CSF, urine, saliva, tears,tissue derived samples, e.g. homogenates, and blood or derivativesthereof.

In many embodiments, the sample is derived from cells that comprise theStat-1 fragments of interest, if present—i.e., if the patient from whichthe cells are derived has chronic immune disease. In other embodiments,the sample may be derived from fluids into which the proteins ofinterest have been released, e.g. are present. In many embodiments, asuitable initial source for the patient sample is blood. As such, thesample employed in the subject assays of these embodiments is generallya blood derived sample. The blood derived sample may be derived fromwhole blood or a fraction thereof, e.g. serum, plasma, etc., where inmany embodiments the sample is derived from blood cells harvested fromwhole blood. Of particular interest as a sample source are mononuclearcells. As such, a preferred sample is one that is derived fromperipheral blood mononuclear cells (PBMCs). In certain situations, thesample may be treated to displace Stat-1 fragments from Stat-1 bindingproteins, where any convenient treatment protocol may be employed, e.g.acidification, etc.

In these preferred embodiments in which the sample is a PBMC derivedsample, the sample is generally a fluid PBMC derived sample. Anyconvenient methodology for producing a fluid PBMC sample may beemployed. In many embodiments, the fluid PBMC derived sample is preparedby: (a) separating PBMCs from whole blood, i.e. collecting PBMCs, e.g.by centrifugation (such as by Ficoll-Hypaque density gradientcentrifugation); (b) disrupting the collected cells, e.g. by contactingwith a lysing buffer; (c) and removing the resultant cellular debris toobtain a cell-free extract, e.g. by centrifugation. A representativemeans for producing a suitable fluid PBMC derived sample, i.e. a fluidPBMC extract, is disclosed in WO 98/15646 and U.S. Pat. No. 5,985,565;the disclosures of which is herein incorporated by reference.

Once the patient derived sample is obtained, it is assayed for thepresence or absence of one or more low molecular weight Stat-1fragments, either directly or indirectly. The low molecular weightStat-1 fragments of interest are those having a molecular weight rangingfrom about 15 to 75 kDa, usually from about 30 to 60 kDa andspecifically of about 45 to 50 kDa, as determined under SDS-PAGEreducing conditions, as described above, with the specific fragment ofinterest being the 50 kDa fragment.

The sample may be assayed for the presence or absence of the lowmolecular weight Stat-1 fragments using any convenient methodology. Inmany embodiments, such methodology involves the following two steps: (a)fractionation of the sample in a manner sufficient such that the one ormore Stat-1 fragments and the native Stat-1 (if present) are present indifferent fractions, i.e. separating the low molecular weight fragmentsfrom each other and from the native Stat-1; and (b) detection of the lowmolecular weight fragments in the specific fractions, i.e. assaying eachfraction for the presence or absence of a Stat-1 fragment, where thedetection may be qualitative, semi-quantitative or quantitative, and isusually at least semi-quantitative (i.e. not just qualitative).

In these embodiments, fractionation may be accomplished using anyconvenient methodology. The fractionation technique employed may or maynot employ native or non-denaturing conditions. Whether fractionation iscarried out under denaturing or non-denaturing conditions depends on theparticular manner in which the low molecular weight fragments aredetected, e.g. whether or not a non-denatured form is required fordetection, where representative detection methods are described ingreater detail below. Typically, the non-denaturing conditions are‘native’ conditions. By ‘native’ conditions is meant fractionation by aprocess that substantially preserves the conformation and folding of thelow molecular fragment species in the sample. Native conditions arethose conditions that do not denature proteins. A variety ofnon-denaturing fractionation means are known to those of skill in theart, where one means of interest is gel filtration high performanceliquid chromatography. Alternatively, fractionation may be carried outunder non-native, e.g. denaturing conditions, such as SDS-PAGE (sodiumdodecylsulfate-polyacrylamide gel electrophoresis). As the fractionatingstep involves separating the various low molecular weight Stat-1fragments, fractionation results in the production of one or morefractions that putatively contain the low molecular Stat-1 fragment(i.e. is suspected of containing a low molecular weight fragment).

As discussed above, the sample or fraction(s) thereof are assayed forthe presence or absence of low molecular weight Stat-1 fragments, wherethe assay may be a direct assay or an indirect assay. By direct assay ismeant an assay that provides for a direct detection of low molecularweight Stat-1 fragments, e.g., an assay that yields direct informationregarding the presence and often amount of low molecular weight Stat-1fragments in a sample, such as an assay where an Stat-1 specificantibody is employed to detect low molecular weight Stat-1 fragments inan appropriately fractionated sample. By indirect assay is meant anassay that detects the presence or absence of low molecular weightStat-1 fragments through detection, usually quantitation, of anotherspecies, e.g. native Stat-1 and total Stat-1 species (e.g., where arelative amount of native Stat-1 to total Stat-1 species in a sample isdetermined, from which the presence of low molecular weight Stat-1fragments is indirectly determined). As such, the assay employed may ormay not also include a determination of the amount of native or fulllength Stat-1, i.e. Stat-1 having a molecular weight of approximately 90kDa in the sample. For example, one can relate an absence of Stat-1native protein to the presence of Stat-1 fragments.

Any convenient assay protocol may be employed. Suitable assays that maybe employed include antibody-based assays, e.g. Western blot assays,such as those described in the experimental section infra. Antibodybased assays require the use of antibodies specific for the Stat-1fragments and/or native Stat-1. The assays may be direct assays, i.e.,those which employ antibodies specific for low molecular weight Stat-1fragments. Alternatively, the assays may be indirect assays, i.e., thosewhich detect native Stat-1 and total amounts of Stat-1 species in asample, e.g., an assay in which an antibodies specific for the C- andN-termini of the native Stat-1 are employed.

Antibodies that specifically bind to the subject Stat-1 protein and lowmolecular weight fragments thereof can be prepared using a variety ofconvenient methods known to those of skill in the art. See Guide toProtein Purification, supra, as well as Antibodies, A Laboratory Manual(Harlow & Lane eds., Cold Spring Harbor Press, 1988). The antibodies maybe polyclonal or monoclonal antibodies depending on the nature of theintended use, as long as they are specific for one or more forms ofStat-1 or fragments thereof of interest.

For preparation of polyclonal antibodies, the first step is immunizationof the host animal with Stat-1 or an immunogenic fragment, includingfragment derivative thereof, where the Stat-1 immunogen will preferablybe in substantially pure form, comprising less than about 1%contaminant. Purified Stat-1 or fragments thereof may be obtained usingany convenient protocol, including that described in U.S. Pat. No.6,030,780, the disclosure of which is herein incorporated by reference.The immunogen may comprise complete Stat-1, fragments or derivativesthereof. To increase the immune response of the host animal, theimmunogen may be combined with an adjuvant, where suitable adjuvantsinclude alum, dextran, sulfate, large polymeric anions, oil & wateremulsions, e.g. Freund's adjuvant, Freund's complete adjuvant, and thelike. The immunogen may also be conjugated to synthetic carrier proteinsor synthetic antigens. A variety of hosts may be immunized to producethe polyclonal antibodies. Such hosts include rabbits, guinea pigs,rodents, e.g. mice, rats, sheep, goats, and the like. The immunogen isadministered to the host, usually intradermally, with an initial dosagefollowed by one or more, usually at least two, additional boosterdosages. Following immunization, the blood from the host is collected,followed by separation of the serum from the blood cells. The Ig presentin the resultant antiserum may be further fractionated using knownmethods, such as ammonium salt fractionation, DEAE chromatography, andthe like.

As with the preparation of polyclonal antibodies, the first step inpreparing monoclonal antibodies specific for Stat-1 and fragmentsthereof is to immunize a suitable host, where suitable hosts includerats, hamsters, mice and the like, and are preferably mice. The Stat-1immunogen, which as above, may be the entire Stat-1 protein or afragment or derivative thereof, is administered to the host in anyconvenient manner, where such methods include: subcutaneous injectionwith adjuvants, nitrocellulose implants comprising the immunogen,intrasplenic injections, and the like, where the immunization protocolmay be modulated to obtain a desired type of antibody, e.g. IgG or IgM,where such methods are known in the art. Following immunization, plasmacells are harvested from the immunized host, where sources of plasmacells include the spleen, lymph nodes and the like, with the spleenbeing preferred. The plasma cells are then immortalized with myelomacells to produce hybridoma cells. A variety of myeloma cell lines areavailable and known to those of skill in the art. The plasma and myelomacells are fused by combining the cells in a fusion medium usually in aratio of about 10 plasma cells to 1 myeloma cell, where suitable fusionmediums include a fusion agent, e.g. PEG 1000, and the like. Followingfusion, the fused cells are selected, e.g. by growing on HAT medium.Following hybridoma cell production, culture supernatant from individualhybridomas is screened for reactivity with Stat-1 using standardtechniques, where such screening techniques include ELISA, dot blotimmunoassays and the like. The antibody may be purified from thesupernatants or ascites fluid by conventional techniques, e.g. affinitychromatography Stat-1 bound to an insoluble support, protein A sepharoseand the like.

Antibodies specific for Stat-1 and fragment thereof are known in theart, and include those specific antibodies described in U.S. Pat. No.6,030,780; the disclosure of which is herein incorporated by reference.

The above prepared or obtained antibodies may be modified in a number ofdifferent ways to optimize their utility for use in a particularimmunoassay. For example, antibody fragments, such as Fv, F(ab)₂ and Fabmay be prepared by cleavage of the intact protein, e.g. by protease orchemical cleavage.

The antibodies, fragments or derivatives thereof may also be labeled inorder to facilitate detection. A variety of protein labeling schemes areknown in the art and may be employed, the particular scheme and labelchosen being the one most convenient for the intended use of theantibody, e.g. immunoassay. Examples of labels include labels thatpermit both the direct and indirect measurement of the presence of theantibody. Examples of labels that permit direct measurement of theantibody include radiolabels, such as ³H or ¹²⁵I, fluorescers, dyes,beads, chemilumninescers, colloidal particles, and the like. Examples oflabels which permit indirect measurement of the presence of the antibodyinclude enzymes where a substrate may provide for a colored orfluorescent product. For example, the antibodies may be labeled with acovalently bound enzyme capable of providing a detectable product signalafter addition of suitable substrate. Instead of covalently binding theenzyme to the antibody, the antibody may be modified to comprise a firstmember of specific binding pair which specifically binds with a secondmember of the specific binding pair that is conjugated to the enzyme,e.g. the antibody may be covalently bound to biotin and the enzymeconjugate to streptavidin. Examples of suitable enzymes for use inconjugates include horseradish peroxidase, alkaline phosphatase, malatedehydrogenase and the like. Where not commercially available, suchantibody-enzyme conjugates are readily produced by techniques known tothose skilled in the art. See also the methods of labeling Stat-1 (andStat-1 fragment) specific antibodies disclosed in U.S. Pat. No.6,030,780; the disclosure of which is herein incorporated by reference.

In immunoassays of the subject invention, a number of differentimmunoassay formats are known in the art and may be employed.Representative assay formats include Western blots on protein gels orprotein spots on filters, where the antibody is labeled as describedabove, as is known in the art. For a representative example of a Westernblot assay for the presence of Stat-1 and fragments thereof in a sample,see the experimental section infra.

Other immunoassays include those based on competitive formats, as areknown in the art. One such format would be where a solid support iscoated with Stat-1. Labeled antibody is then combined with the patientderived sample suspected to produce a reaction mixture which, followingsufficient incubation time for binding complexes to form, is contactedwith the solid phase bound Stat-1. The amount of labeled antibody whichbinds to the solid phase will be proportional to the amount of Stat-1 orfragments thereof in the sample, and the presence of Stat-1 andfragments thereof may therefore be detected. Other competitive formatsthat may be employed include those where the sample suspected ofcomprising Stat-1 fragments is combined with a known amount of labeledStat-1 fragments and then contacted with a solid support coated withantibody specific for Stat-1 fragments. Such assay formats are known inthe art and further described in both Guide to Protein Purification,supra, and Antibodies, A Laboratory Manual, supra. Sandwich-formatassays may also be employed. A sandwich assay is performed by initiallyattaching a first of the two types of antibodies to an insoluble surfaceor support. This first antibody may be bound to the surface by anyconvenient means, depending upon the nature of the surface, eitherdirectly or through specific antibodies. The particular manner ofbinding is not crucial so long as it is compatible with the reagents andoverall methods of the invention. They may be bound to the platescovalently or non-covalently, preferably non-covalently. The insolublesupports may be any compositions to which antibodies or fragmentsthereof can be bound, which is readily separated from soluble material,and which is otherwise compatible with the overall method of measuringStat-1 in the sample. The surface of such supports may be solid orporous and of any convenient shape. Examples of suitable insolublesupports to which the receptor is bound include beads, e.g. magneticbeads, membranes and microtiter plates. These are typically made ofglass, plastic (e.g. polystyrene), polysaccharides, nylon ornitrocellulose. Microtiter plates are especially convenient because alarge number of assays can be carried out simultaneously, using smallamounts of reagents and samples. Before adding patient samples orfractions thereof, the non-specific binding sites on the insolublesupport i.e. those not occupied by the first antibody, are generallyblocked. Preferred blocking agents include non-interfering proteins suchas bovine serum albumin, casein, gelatin, and the like. Alternatively,several detergents at non-interfering concentrations, such as Tween,NP40, TX100, and the like may be used. Samples, fractions or aliquotsthereof are then added to separately assayable supports (for example,separate wells of a microtiter plate) containing support-bound allergen.Preferably, a series of standards, containing known concentrations ofStat-1 is assayed in parallel with the samples or aliquots thereof toserve as controls. Generally from about 0.001 to 1 ml of sample, dilutedor otherwise, is sufficient, usually about 0.01 ml sufficing.Preferably, each sample and standard will be added to multiple wells sothat mean values can be obtained for each. The incubation time should besufficient for Stat-1 molecules to bind the insoluble first antibody.Generally, from about 0.1 to 3 hr is sufficient, usually 1 hr sufficing.After incubation, the insoluble support is generally washed of non-boundcomponents. Generally, a dilute non-ionic detergent medium at anappropriate pH, generally 7-8, is used as a wash medium. From one to sixwashes may be employed, with sufficient volume to thoroughly washnon-specifically bound proteins present in the sample. After washing, asolution containing the second Stat-1 or Stat-1 fragment specificantibody is applied. The second antibody may be labeled, as describedabove, to facilitate direct, or indirect detection and/or quantificationof binding. Examples of labels which permit direct measurement ofimmunocomplexes include radiolabels, such as ³H or ¹²⁵I, fluorescers,dyes, beads, chemilumninescers, colloidal particles, and the like.Examples of labels which permit indirect measurement of binding includeenzymes where the substrate may provide for a colored or fluorescentproduct. In a preferred embodiment, the second antibody is labeled witha covalently bound enzyme capable of providing a detectable productsignal after addition of suitable substrate. Examples of suitableenzymes for use in conjugates include horseradish peroxidase, alkalinephosphatase, malate dehydrogenase and the like. Where not commerciallyavailable, such antibody-enzyme conjugates are readily produced bytechniques known to those skilled in the art. Alternatively, theantibody may be unlabeled. In this case, a labeled secondreceptor-specific compound is employed which binds to the secondantibody. Such a second receptor-specific compound can be labeled in anyof the above manners. It is possible to select such compounds such thatmultiple compounds bind each molecule of bound second receptor. Examplesof second antibody/second receptor-specific molecule pairs includeantibody/anti-antibody and avidin (or streptavidin)/biotin. Since theresultant signal is thus amplified, this technique may be advantageouswhere only a small amount of Stat-1 or fragment thereof is present. Anexample is the use of a labeled antibody specific to the secondantibody. The volume, composition and concentration of second antibodysolution provides for measurable binding to the Stat-1 already bound tothe first antibody. Generally, the same volume as that of the sample isused: from about 0.001 to 1 ml is sufficient, usually about 0.1 mlsufficing. The concentration will generally be sufficient to saturateall Stat-1 potentially bound to first antibody. The concentrationgenerally will be about 0.1 to 50 μg/ml, preferably about 1 μg/ml. Thesolution containing the second antibody is generally buffered in therange of about pH 6.5-9.5. The solution may also contain an innocuousprotein as previously described. The incubation time should besufficient for the labeled ligand to bind available molecules.Generally, from about 0.1 to 3 hr is sufficient, usually 1 hr sufficing.After the second antibody has bound, the insoluble support is generallyagain washed free of non-specifically bound second receptor, essentiallyas described for prior washes. After non-specifically bound material hasbeen cleared, the signal produced by the bound conjugate is detected byconventional means. Where an enzyme conjugate is used, an appropriateenzyme substrate is provided so a detectable product is formed. Morespecifically, where a peroxidase is the selected enzyme conjugate, apreferred substrate combination is H₂O₂ and O-phenylenediamine whichyields a colored product under appropriate reaction conditions.Appropriate substrates for other enzyme conjugates such as thosedisclosed above are known to those skilled in the art. Suitable reactionconditions as well as means for detecting the various useful conjugatesor their products are also known to those skilled in the art. For theproduct of the substrate O-phenylenediamine for example, lightabsorbance at 490-495 nm is conveniently measured with aspectrophotometer.

Depending on the particular nature of the antibody based assay employed,it may be desirable to employ antibodies that are capable ofdistinguishing between the various Stat-1 forms and fragments thereof.For example, in a Western blot assay a single type of antibody thatrecognizes all of the various Stat-1 fragments and the native Stat-1itself may be employed, since the various fragments and native proteinare pre-separated, e.g. by gel electrophoresis. However, where thevarious fragments and native protein are not separated prior todetection, e.g. in the competitive and sandwich assays described above,it is desirable to use a plurality of antibodies which are capable ofspecifically recognizing only a single Stat-1 species of interest, withsubstantially no cross-reactivity with other Stat-1 species or fragmentsthat may be present in the sample.

In the subject methods, the sample or fractions thereof are at leastassayed for the presence or absence of the low molecular Stat-1fragments or species, and often times the native species as well, wherethe assay may be a direct assay for low molecular weight fragments or anindirect assay for low molecular weight fragments, as indicated above.In some embodiments, qualitative results are sufficient. Thus, one maybe interested in identifying the presence or absence of the lowmolecular weight Stat-1 fragments as a marker for the chronic immunedisease, e.g. in the diagnostic methods described above. Alternatively,one may be interested in making a qualitative determination of the ratioof the low molecular weight species to the native species. In manyembodiments, the assays employed at least provide semi-quantitativedetection of the various molecular weight Stat-1 species, and not justqualitative detection.

In assaying for low molecular weight Stat-1 fragments or species in thesubject methods, one may look for: (a) the presence or absence of thelow molecular weight fragments; (b) the pattern of the low molecularweight fragments and, optionally full length Stat-1 (where by pattern ismeant the presence of each fragment and, optionally relative amount ofeach fragment); (c) the ratio of the amounts of the various lowmolecular weight species to each other and/or to the full length Stat-1;and the like; (d) the relative amount of high molecular weight or nativeStat-1 to all Stat-1 species in the sample; etc.

In many embodiments, based on the presence or absence of the variousmolecular weight Stat-1 species, and usually the semi-quantitativevalues obtained for each of the species of interest, the chronic immunedisease activity in the subject from which the sample was derived ischaracterized. This broad category of embodiments includes those inwhich the low molecular weight Stat-1 species are directly assayed,e.g., those methods where: (a) the simple presence or absence of lowmolecular weight species is used to characterize the disease; (b) theratio of low molecular weight species to high molecular weight speciesis used to characterize the disease; and (c) the pattern or amounts oftwo or more different low molecular weight species is used tocharacterize the disease; etc.

In yet other embodiments, e.g. those based on assays which indirectlydetermine the presence or absence of low molecular weight Stat-1species, the relative amounts of the various Stat-1 species in thesample to each other, e.g., the relative amount of native or highmolecular weight Stat-1 to the total amount of Stat-1, i.e., nativeStat-1 and fragment species thereof, in the sample is used tocharacterize the chronic immune disease activity in the subject.

Characterization of chronic immune disease activity according to thesubject methods typically involves comparing the results obtained to atable or other source of predetermined values or reference values whichprovide information about the disease activity in the host, e.g. thatpositively or negatively correlate to the presence of the chronic immunedisease, a particular stage of the chronic immune disease, and the like.For example, a table of values may be consulted in this step, where thetable comprises representative values for the high and low molecularweight proteins as found in patients suffering from the chronic immunedisease of interest. The values may be presented in numerical form, inpicture form (e.g. as bands on a gel), and the like. By comparing theobserved values with these reference values, e.g. by comparing a patternof the Stat-1 species in the sample to a reference pattern or picture,characterization of the disease activity, e.g. confirmation ofdiagnosis, determination of disease state, etc., is readily made. Inother embodiments, the ratio of two or more of the different speciesand/or full length Stat-1 is then compared to reference list of ratiosto characterize the chronic immune disease activity.

As summarized above, the subject methods are methods of characterizingchronic immune disease activity in a host. The term characterizing isused broadly to refer to derivation of any type of information about thestate of the chronic immune disease in the host. As such, the subjectmethods may be used to confirm an initial diagnosis of chronic immunedisease, to determine the state of the disease in a patient known tohave the chronic immune disease, to monitor the progression of thedisease, to predict the occurrence of an attack, and the like. Where thesubject invention is employed to confirm an initial diagnosis, a sampleis obtained from subject suspected of having the chronic immune disease(where the subject may be identified as described supra). For example,the sample is assayed for the presence of the high and low molecularweight Stat-1 species, a ratio of the two species is derived and thencompared to reference values, where the reference values correlate givenratios to the presence or absence of the chronic immune disease.

The subject methods are also employed to determine the stage of thechronic immune disease in the subject. In other words, the subjectchronic immune disease activity characterization methods may be employedto determine whether the patient is in a remission stage, a chronicstage etc. For example, the subject methods may be employed to determinewhether an MS patient is in the relapsing-remitting stage or in thechronic progressive stage of the disease. To determine the stage of thedisease, the observed values for the one or more Stat-1 species, andratios where desired, in the assayed sample are compared to referencevalues that are correlated to a particular stage of chronic immunedisease, e.g. remitting relapsing or chronic progressive stage of MS.

In yet other embodiments, characterization of disease activity yieldsinformation concerning the disease progression in the patient, e.g.whether disease progression has accelerated or slowed. For example, theinitial characterization date, i.e. the amount of high (i.e., native)and low molecular weight forms of Stat-1 in the patient derived samplecould be employed as a baseline value to evaluate subsequent testings,e.g. at some time following the initial testing, e.g. 3 months. If theamount of low molecular weight form decreases in subsequent testing,this indicates that the disease is not progressing. Alternatively, ifthe amount of low molecular weight form increases, this indicates thatthe disease is progressing in severity.

The characterization data obtained from the subject methods may also beused to determine whether a particular therapeutic regimen is havingpositive affects with respect to the progression of the disease. Forexample, at various time periods during the course of treatment, thesubject methods may be performed to obtain a reading of the amount ofhigh and low molecular weight forms of the Stat-1 species of interest.If the amount of the low molecular weight marker is increasing, thisindicates that the treatment regimen is not having the desired effect.Alternatively, if the amount of the low molecular weight marker isdecreasing, this indicates that the treatment regimen is working.

In yet other embodiments, the characterization data obtained from thesubject methods is used to predict when a chronic immune disease attack,e.g. MS attack, may occur. In this embodiment, the characterization datais compared to reference values, where some of the reference valuescorrelate to the occurrence of an attack.

Depending on the particular test protocol, the subject methods mayfurther include one or more additional assays associated with thechronic immune disease of interest. For example, one may couple thesubject methods with assays that look for the presence of low molecularweight proteins that exhibit RNase L activity, the ratio of high to lowmolecular weight proteins that exhibit RNase L activity, etc., asdescribed in U.S. Pat. Nos. 5,985,565; 6,080,554; 6,207,366; and6,214,544 the disclosures of which are herein incorporated by reference.Other representative assays of interest include biochemical assayscapable of identifying MS activity in the subject, e.g. assays whichdetect the presence of oligoclonal bands in cerebral spinal fluid (CSF).A variety of such assays are known to those of skill in the art and maybe employed in the subject methods. See e.g. Mehta et al.,Electrophoresis 9:126-8, 1998; Mehta et al., J Clin Lab Immunol.6:17-22, 1981; Trbojevic-Cepe et al., Neurologija. 38:11-21, 1989; Lasneet al., J. Neurochem. 36:1872-4, 1981; Mehta et al., J Neurosci Methods16:277-82, 1986.

Also provided by the subject invention are kits for use in carrying outthe subject methods. The kits at least comprise reagents necessary forcarrying out the Stat-1 species detection assays, where such kits mayinclude: Stat-1 specific antibodies and/or immunoassay devicescomprising the same; members of a signal producing system, such asantibodies, enzyme substrates, and the like; various buffers for use incarrying out the subject detection assays; and the like. The kits mayfurther include one or more reagents necessary for preparation of thepatient derived sample, such as heparin, Ficoll-Hypaque, lysing buffer,protease inhibitor, and the like, e.g. where the patient sample is PBMCderived, etc. In addition, the subject kits may further include one ormore components employed in fractionation of the sample, such as anelectrophoretic medium or precursors thereof, e.g. dried precursors ofpolacrylamide gels, one or more buffer mediums or components thereof,and the like. In most embodiments, the kits further include at least aninformation storage and presentation medium that contains reference datawith which assay results may be compared in order to diagnose and/orcharacterize the chronic immune disease activity in the subject beingassayed, i.e. reference data that includes various values of the highand low molecular weight Stat-1 species and relates these values to thepresence or absence of chronic immune disease and/or the activity of thedisease in the host. The information storage and presentation medium maybe in any convenient form, such as a printed information on a packageinsert, an electronic file present on an electronic storage medium, e.g.a magnetic disk, CD-ROM, and the like. In yet other embodiments, thekits may include alternative means for obtaining reference data, e.g. awebsite for obtaining the reference data “on-line.” The kits may furtherinclude means for obtaining the patient sample, e.g. a syringe. Thesubject kits further typically include instructions for carrying out thesubject methods, where these instructions may be present on a packageinsert and/or the packaging of the kit. Finally, the kit may furtherinclude one or more reagents from an additional biochemical assay whichis used to detect the presence of and/or characterize the chronic immunedisease of interest. For example, where MS is the chronic immune diseaseof interest, the kits may further include one or more reagents from anassay designed to detect the presence of oligoclonal bands in CSF, e.g.immunoxification reagents (e.g. anti-IgG); labeling reagents, such assilver salts, and the like.

Also provided by the subject invention are assay methods for use indetecting the proteolytic activity of a sample with respect to directcleavage of native Stat-1 protein and/or recombinant Stat-1 protein. Inthese assay methods of the subject invention, a subject sample, asdescribed above, is contacted with a source of native and/or recombinantStat-1 protein under conditions sufficient for Stat-1 protein cleavageproducts to be generated if the sample comprises the proteolyticactivity of interest. Generally, contact is maintained for a period oftime sufficient for a representative amount of cleavage products to beproduced, where this incubation time typically ranges from about 5 to120 minutes, usually from about 30 to 60 minutes. The source of nativeStat-1 protein and/or recombinant Stat-1 protein that may be used inthese assays may be any convenient source. As such, the source may be anaturally occurring source, a recombinant source and the like.

Any convenient cleavage product detection format may be employed.Depending on the detection format employed, the source of native and/orrecombinant Stat-1 protein may or may not be labeled. For example, oneconvenient assay employs the use of substrate bound native and/orrecombinant Stat-1, where the proteins are labeled, generally proximalto or at the end of the protein that is not attached, either directly orindirectly, to the substrate. The substrate bound protein is thencontacted with the sample, as described above, and, followingincubation, any cleavage products, e.g. low molecular weight Stat-1cleavage products, are detected. Non-labeled protocols may also beemployed, e.g. antibody based (such as Western blot formats) asdescribed supra.

Following detection of the cleavage products, the presence of, andgenerally amount of cleavage products is related to the proteolyticactivity of the sample, specifically the Stat-1 proteolytic activity ofthe sample. In other words, the pattern of native and/or recombinantStat-1 cleavage products or proteins in the sample is related to theproteolytic characteristics or ability of the sample. For example, thepresence of cleavage products indicates that the sample comprises thetarget proteolytic activity, while the amount of the cleavage productsindicates the level of proteolytic activity.

The above assay for proteolytic activity in the sample may be employedin many applications. For example, the above proteolytic activity assaymay be employed in addition to, or as a substitute for, the Stat-1species detection assays in the above described methods of diagnosingand/or characterizing chronic immune disease activity.

Also provided are kits for use in practicing the subject proteolyticactivity assays. The subject kits include, among other components, asource of native and/or recombinant Stat-1 (e.g. source of full lengthrecombinant Stat-1 protein), where the source may be stably associatedwith the surface of a substrate and/or labeled, depending on the natureof the assay to be performed. Generally, the kits will also comprise amedium having reference values recorded thereon for use in interpretingthe assay data and relating the data to the proteolytic activity in thesample.

In addition to the diagnostic/characterization applications describedabove, the above described methods also find use in evaluating theeffectiveness of a particular chronic immune disease therapeuticprotocol. More specifically, also provided are methods of evaluatingwhether or not interferon based therapy for chronic immune disease willbe effective for a given subject, i.e., whether a particular subjectsuffering from chronic immune disease will benefit from interferon basedtherapy (i.e., therapy with interferon or inducers thereof, as describedabove).

In such methods, the first step is to determine the amount of native orfull length Stat-1 in the relevant patient sample, e.g., PBMC, asdescribed above. Based on the amount of native Stat-1 present, thedecision is made as to whether or not interferon-based therapy isindicated. More specifically, native Stat-1 present in the sampleindicates that interferon based therapy will be effective, while anabsence of Stat-1 present in the sample indicates that Stat-1 therapywill not be effective. As such, following detection of the presence orabsence of native Stat-1 in the sample, a decision is made as to whetheror not interferon based therapy will be effective.

As summarized above, the subject invention also provides methods fortreating a host suffering from a chronic immune disease. Specifically,the subject invention provides methods of treating a host suffering fromMS or CFS.

In practicing the subject methods, an effective amount of an agent thatenhances Stat-1 protein activity, specifically in PBMC, is administeredto the host suffering from the chronic immune disease. By enhance ismeant that the Stat-1 activity in the host, particularly in PBMC of thehost, is increased by at least about 2 fold, usually by at least about 3fold and more usually by at least about 5 fold, as compared to thatobserved in a control, i.e., a PBMC from the host that has not beencontacted by the active agent(s).

Enhancement of Stat-1 activity can be accomplished in any convenientmanner. Particular active agents of interest include, but are notlimited to Stat-1 cleavage-inhibitory agents and Stat-1 expressionenhancing agents. Each of these types of agents is now describedseparately in greater detail.

Stat-1 Cleavage-Inhibitory Agents

Stat-1 cleavage-inhibitory agents of interest for use in the subjectmethods are agents that inhibit cleavage or fragmentation of Stat-1protein. The target molecule is a protein or activity, e.g., an enzymethat cleaves native Stat-1 protein into fragments. An example of such aprotein with Stat-1 cleavage ability is caspase 3 (see King et al., J.Biol. Chem. 273:8699-8704, 1998). By inhibit is meant that these agentsat least reduce, if not substantially or completely stop, the cleavageof Stat-1. Stat-1 cleavage-inhibitory agents typically reduce thecleavage or Stat-1 by at least about 2 fold, usually at least about 3fold and more usually at least about 5 fold. Inhibitors of interestinclude agents that bind to the target molecule (e.g., protease) andconcomitantly reduce its activity, as well as agents that reduce theexpression of the target molecule so that the overall cleavage activityof the target molecule is reduced. As such, agents of interest includesmall molecule agents, as may be identified in the assays describedbelow and antibodies specific to inhibiting the action of the Stat-1cleaving target molecules. Small molecule agents of interest includesmall organic compounds having a molecular weight of more than 50 andless than about 2,500 daltons. The small molecule agents comprisefunctional groups necessary for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. The small molecule agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Small molecule agents of interest are also foundamong biomolecules including, but not limited to: peptides, saccharides,fatty acids, steroids, purines, pyrimidines, derivatives, structuralanalogs or combinations thereof. Candidate agents are obtained from awide variety of sources including libraries of synthetic or naturalcompounds. For example, numerous means are available for random anddirected synthesis of a wide variety of organic compounds andbiomolecules, including expression of randomized oligonucleotides andoligopeptides. Alternatively, libraries of natural compounds in the formof bacterial, fungal, plant and animal extracts are available or readilyproduced. Additionally, natural or synthetically produced libraries andcompounds are readily modified through conventional chemical, physicaland biochemical means, and may be used to produce combinatoriallibraries. Known pharmacological agents may be subjected to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs. Newpotential therapeutic agents may also be created using methods such asrational drug design or computer modeling. Protease specific antibodiesmay be readily produced using the procedures described above.

In yet other embodiments of the invention, the active agent is an agentthat modulates, and generally decreases or down regulates, theexpression of the Stat-1-specific protease gene in the host. Antisensemolecules can be used to down-regulate expression of genes in cells. Theantisense reagent may be antisense oligonucleotides (ODN), particularlysynthetic ODN having chemical modifications from native nucleic acids,or nucleic acid constructs that express such anti-sense molecules asRNA. The antisense sequence is complementary to the mRNA of the targetedgene, and inhibits expression of the targeted gene products. Antisensemolecules inhibit gene expression through various mechanisms, e.g. byreducing the amount of mRNA available for translation, throughactivation of RNAse H, or steric hindrance. One or a combination ofantisense molecules may be administered, where a combination maycomprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal., Nature Biotechnol. 14:840-844, 1996).

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al., supra, and Milligan et al., supra.)Preferred oligonucleotides are chemically modified from the nativephosphodiester structure, in order to increase their intracellularstability and binding affinity. A number of such modifications have beendescribed in the literature to alter the chemistry of the backbone,sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-0′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH₂-5′-O-phosphonate and 3′—NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g. ribozymes, anti-sense conjugates, etc. may be used toinhibit gene expression.

Ribozymes may be synthesized in vitro and administered to the patient,or may be encoded on an expression vector, from which the ribozyme issynthesized in the targeted cell (for example, see International patentapplication WO 9523225, and Beigelman et al., Nucl. Acids Res.23:4434-42, 1995). Examples of oligonucleotides with catalytic activityare described in WO 9506764. Conjugates of anti-sense ODN with a metalcomplex, e.g. terpyridylCu(II), capable of mediating mRNA hydrolysis aredescribed in Bashkin et al., Appl. Biochem. Biotechnol. 54:43-56, 1995.

A further alternative to the above is the use of double-stranded RNAsequences, or the production thereof by introducing vectors for such inthe host, the nucleic acid sequences of which are identical to all orpart of the Stat-1-specific protease gene. Such a double-stranded RNA iscapable of binding to and causing the degradation of the homologous mRNAspecies. Thus, the mRNA coding for the production of Stat-1-specificprotease is targeted for removal by this method. This technique isreferred to as RNA interference, examples of which are described inTuschl et al., Genes and Development 13:3191-3197, 1999, and Zamore,Cell 101:25-33, 2000.

Stat-1 Expression Enhancing Agents

In yet other embodiments of the subject invention, the active agent is aStat-1 expression-enhancing agent. By Stat-1 expression enhancing agentis meant an agent that enhances expression of native Stat-1 mRNA and/orthe production of native Stat-1 protein in the host, particularly inPBMC of the host. Agents of interest include, but are not limited to:Stat-1 nucleic acid and protein therapeutic compositions. In thisembodiment, the genes or gene fragments are useful in gene therapy toenhance Stat-1 gene activity. Expression vectors may be used tointroduce the Stat-1 gene into a cell. Such vectors generally haveconvenient restriction sites located near the promoter sequence toprovide for the insertion of nucleic acid sequences. Transcriptioncassettes may be prepared comprising a transcription initiation region,the target gene or fragment thereof, and a transcriptional terminationregion. The transcription cassettes may be introduced into a variety ofvectors, e.g., plasmid; retrovirus, e.g. lentivirus; adenovirus; and thelike, where the vectors are able to transiently or stably be maintainedin the cells, usually for a period of at least about one day, moreusually for a period of at least about several days to several weeks.

The gene or protein may be introduced into tissues or host cells by anynumber of routes, including viral infection, microinjection, or fusionof vesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al., Anal. Biochem.205:365-368, 1992. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”as described by Tang et al., Nature 356:152-154, 1992, where goldmicroprojectiles are coated with the DNA and then bombarded into skincells. The nucleic acid and protein sequence of Stat-1 is known, wherethe human mRNA and amino acid sequence are deposited in GenBank underAccession no. NM_(—)007315.

Also of interest is the use of agents that modulate the endogenousStat-1 gene of the host to enhance its expression. For example, theendogenous Stat-1 gene of a cell can be regulated by an exogenousregulatory sequence inserted into the genome of the cell at locationsufficient to at least enhance expressed of the gene in the cell. Theregulatory sequence may be designed to integrate into the genome viahomologous recombination, as disclosed in U.S. Pat. Nos. 5,641,670 and5,733,761, the disclosures of which are herein incorporated byreference, or may be designed to integrate into the genome vianon-homologous recombination, as described in WO 99/15650, thedisclosure of which is herein incorporated by reference. As such, alsoencompassed in the subject invention is the enhancement of Stat-1expression without manipulation of the encoding nucleic acid itself, butinstead through integration of a regulatory sequence into the genome ofcell of the host that already includes a gene encoding the desiredprotein, as described in the above incorporated patent documents.

Also of interest is the use of agents that modulate the levels of nativeStat-1 protein in the host, particularly in PBMC of the host. Such anagent may act directly on the PBMCs of the host, such as one of theinterferon class of proteins, or indirectly on the PBMCs through theinduction of interferon proteins (see Bottrel et al., Antimicrob. AgentsChemother. 43:856-861, 1999). In such instance where the agent acts toinduce the production of interferon, the combination of enhancedexpression of Stat-1 protein via gene therapy techniques and theinduction of interferon through the administration of such agents asimidazoquinolines, may result in significant therapeutic benefit to thepatient.

As mentioned above, in the subject methods an effective amount of one ormore of the above described active agents is administered to the host,where “effective amount” means a dosage sufficient to produce a desiredresult, where the desired result is at least an amelioration, if notcomplete cessation of the chronic immune disease symptoms.

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredtreatment. Thus, the agent can be incorporated into a variety offormulations for therapeutic administration. More particularly, theagents of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants and aerosols.

As such, administration of the agents can be achieved in various ways,including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Where the agent is a polypeptide, polynucleotide, analog or mimeticthereof, e.g. antisense composition, it may be introduced into tissuesor host cells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al., supra.The agent may be coated onto gold microparticles, and deliveredintradermally by a particle bombardment device, or “gene gun” asdescribed in the literature as described by Tang et al., supra.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

As mentioned above, by treatment is meant that at least an ameliorationof the symptoms associated with the chronic immune disease, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thecondition being treated. As such, treatment also includes situationswhere the pathological condition, or at least symptoms associatedtherewith, are completely inhibited, e.g. prevented from happening, orstopped, e.g. terminated, such that the host no longer suffers from thecondition, or at least the symptoms that characterize the chronic immunedisease condition.

In treating subjects according to the subject invention, the Stat-1activity enhancing agent may be administered by itself, or inconjunction with one or more additional chronic immune diseasetherapeutic agents. For example, the Stat-1 activity enhancing agent maybe administered in conjunction with an interferon agent, e.g.,interferon or an inducer thereof, as described above and in U.S. Pat.No. 6,013,253, the disclosure of which is herein incorporated byreference.

A variety of hosts are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the hosts will be humans.

Kits with unit doses of the active agent, usually in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental Section

Venous blood was collected from patients with Chronic Fatigue Syndrome,Multiple Sclerosis, and from healthy controls using standard steriletechniques. Patients with CFS as diagnosed by meeting the establishedcase definition were selected from a special clinic at the FreeUniversity of Brussels, Brussels, Belgium. Patients with MS as diagnosedby standard procedures were selected from a special clinic in Overpelt,Belgium. Blood from healthy volunteer controls was provided by theSports Medicine center at the Free University of Brussels, Brussels,Belgium. Patient consent was obtained in all cases.

I. Procedures A. Cell Isolation and Protein Extraction

Peripheral blood mononuclear cells (PBMCs) were separated fromheparinized blood (30 mLs) by Ficoll-Hypaque density gradientcentrifugation. The blood was layered onto 20 mLs of Ficoll-Hypaque(Boyum, Scandinavian Journal of Clinical Laboratory Investigation,97:101-109, 1968) at a density of 1.077 g/mL at 20 C and centrifuged for30 minutes at 500×g. The PBMC layer was removed and washed once with 5volumes of phosphate buffered saline (PBS). The cells were thenresuspended in 5 mLs of red blood cell lysing buffer (155 mM NH₄Cl, 10mM NaHCO₃, 0.1 mM EDTA, pH 7.4), kept on ice for 5 minutes, thencentrifuged for 5 minutes at 500×g. The resultant cell pellet was washedonce with 15 mLs of PBS and centrifuged for 5 minutes at 500×g. Theresultant pellet was then stored at −70 C until the protein extractionprocedure could be performed.

To extract the proteins from the cell pellet, PBMCs were resuspended ina volume approximately 5-10 times the packed cell volume in the extractbuffer (10 mM HEPES, pH 7.6, 90 mM KCl, 1.5 mM Mg(OAc)₂, 0.5% non-ionicdetergent (such as Nonidet P-40 or Igepal CA-630, Sigma ChemicalCorporation)). The extract buffer also contained a mixture of proteaseinhibitors to help stabilize the extract and impeded the action ofproteases. Once such commercially available mixture is the MiniCompleteprotease inhibitor cocktail (Boehringer-Mannheim). This containsaprotinin, leupeptin, pefabloc-SC and EDTA.

The extraction procedure was performed at 2-4 degrees C., holding thecell pellet-extraction buffer in ice water or on wet ice for 5 minutes.The cell pellet-buffer mix was then vortexed at medium speed for 2minutes at room temperature to ensure complete solubilization of thecell membranes. The cell pellet-buffer mix was then placed at 2-4 C foran additional 5 minutes. The final step was to centrifuge the cellpellet-buffer mix at high speed in a microcentrifuge (16,000×g) for 2minutes. The supernatant containing the proteins of interest wascollected and the cell pellet is discarded. All cell extracts werestored at −70 C until further analysis could be performed.

Quantification of protein in the patient cell extracts was performedusing a standard commercially available procedure of a modified Bradfordmethod (Bio-Rad Laboratories) following the manufacturer's recommendedprocedure.

B. Quantification of 2′-5′A Binding Proteins

Analysis of LMW and HMW 2′5′A binding proteins was performed using aradiolabeled 2′-5′A trimer and SDS-PAGE as described by the method ofCharachon et al. (Biochemistry 29:2550-2556, 1990). Briefly, 2′-5′Atrimer was radiolabeled by the ligation of ³²P-pCp to the 3′ end (methodof Charachon). After removal of the 3′ terminal phosphate by treatmentwith bacterial alkaline phosphatase, the 3′ ribose residue of pC wasoxidized with sodium metaperiodate (10 mM final concentration, pH 4.75)for one hour at 4 C to form 2′5′A-³² pC-OX. This reaction mixture wassubsequently equilibrated to pH 8.0 by the addition of NaOH. Thisoxidized molecule was used as the radiolabel in all subsequent reactionsfor RNase L protein analysis (referred to below as radiolabeled 2′5′A).

The radiolabeled 2′5′A was incubated with 200 micrograms of cell extractat 2-4 C for 15 minutes to allow the radiolabeled 2′5′A to interact withany 2′5′A-binding proteins present, such as RNase L (all molecularweight species). The 2′-5′A radiolabel was then covalently attached toall RNase L species by the addition of cyanoborohydride (20 mM in 100 mMphosphate buffer, pH 8.0). The reduction reaction was allowed to occurfor 20 minutes at room temperature. SDS-PAGE sample buffer, including atracking dye, was added to the samples and all samples were incubated at95 C for 5 minutes to reduce any disulfide bonds present.

The samples were then subjected to standard SDS-polyacrylamide gelelectrophoresis using a 4 percent stacking gel and a 10 percentseparating gel (Bisbal et al, European Journal of Biochemistry179:595-602, 1989). Also included in the first lane of each gel was amolecular weight marker, pre-stained to be visible as it migrated duringthe course of electrophoresis (Bio-Rad Laboratories). The gel waselectrophoresed until the tracking dye had migrated to the bottom of thegel (approximately 5 hours at a constant current of 30 mAmps). The gelwas then dried and subjected to autoradiography (Bio-Rad Laboratories FXImager).

The autoradiographs were then analyzed by densitometry, andquantification of any and all RNase L species present was performedusing specialized software (Quantity One from Bio-Rad Laboratories). Theresults are expressed as the density (or relative amount) of 37 kDa LMWRNase L present divided by the density (or relative amount) of 80 kDaHMW RNase L present, multiplied by a constant factor of 10.

C. Quantification of Stat-1 Protein and Related Fragments by WesternBlot

Briefly, the procedure used is as follows: 200 micrograms of proteinextracted from the cytoplasm of PBMCs was mixed with 2×SDS-PAGE gelsample dye that included a tracking dye, and heated to 95 C for fiveminutes to denature the proteins. The denatured samples were thensubjected to standard SDS-PAGE using a 4 percent stacking gel and 10percent separating gel. Also included in the first lane of each gel wasa molecular weight marker, pre-stained to be visible as it migratedduring the course of electrophoresis (Bio-Rad Laboratories). The gel waselectrophoresed until the tracking dye had migrated to the bottom of thegel.

The gel was then transferred to a PVDF membrane (Bio-Rad Laboratories)using a semi-dry transfer system (Amersham-Pharmacia Biotech). Transferwas performed at an average current of 0.8 milliamp per cm2 of gel (or100 mA for a standard 15 cm×8 cm gel) for two hours. After transfer wascomplete (as determined by the visual agreement of the transfer of thecolor from the pre-stained molecular weight markers to the membrane),the membrane was allowed to dry thoroughly at room temperature for atleast one hour.

Western blotting was performed using the following format: The membranewas first wet with a minimum volume of 100 percent methanol (accordingto the manufacturer's instruction). Then a solution of five percentnon-fat dry milk (5% NFDM) was used to ‘block’ the membrane (‘blockingbuffer’) to eliminate non-specific background binding of antibody. Themembrane was ‘blocked’ for one hour with gentle shaking on an orbitalshaker.

The blocking buffer was discarded and fresh blocking buffer was added inthe amount of approximately 0.1 mL per cm2 of membrane, to which wasadded the primary antibody (mouse anti-Stat-1 monoclonal antibody; SantaCruz Biotechnologies, catalog number sc-417) at a 1:100 dilution. Themembrane was allowed to react with the primary antibody for one hourwith gentle shaking on an orbital shaker. The primary antibody solutionwas then discarded and the membrane was washed three times with 25 mLsper wash of phosphate buffered saline (PBS, pH=7.4) plus 0.1% Tween 20(polyoxyethylene sorbitan monolaurate; Sigma Corporation). Each wash wasfive minutes in length, with shaking, and the each time the solution wasdiscarded.

Fresh blocking buffer was added in the amount of approximately 0.1 mLper cm2 of membrane, to which was added the secondary antibody (goatanti-mouse antibody, conjugated to horseradish peroxidase (GAR-HRP);Bio-Rad Laboratories) at a 1:2000 dilution according to themanufacturer's recommendations. The membrane was allowed to react withthe secondary antibody for thirty minutes with gentle shaking on anorbital shaker. The secondary antibody solution was discarded and themembrane was washed three times with 25 mLs per wash of phosphatebuffered saline (PBS, pH=7.4) plus 0.1% Tween 20. Each wash was fiveminutes in length, with shaking, and the each time the solution wasdiscarded.

Color development was performed using the Opti4-CN kit (Bio-RadLaboratories) according to the manufacturer's recommendations. Colordevelopment was allowed to proceed for 15 minutes and the membrane wasthen rinsed in copious changes of water and allowed to dry at roomtemperature. The membrane was then analyzed by densitometry andquantification of Stat-1 and Stat-1 fragment proteins present wasperformed using specialized software (Quantity One from Bio-RadLaboratories).

As a control to ensure that the disappearance of Stat-1 protein was notdue to non-specific proteolysis resulting in the degradation of allproteins present, Western blotting was performed with anti-Bcl-2antibody (mouse anti-Bcl-2 monoclonal antibody; Santa CruzBiotechnologies, catalog number sc-509) at a 1:100 dilution. The amountof PBMC protein extract used and the conditions for SDS-PAGE, transferto PVDF membrane, Western blotting, color development and densitometrywere performed exactly as described above.

D. Effect of Protease Inhibitors on Stat-1 Protein Degradation

To demonstrate the presence of Stat-1-specific proteases in PBMCextracts, two extracts were mixed in the presence or absence of specificprotease inhibitors. One of the two extracts used was from a healthycontrol (i.e., RNase L ratio<0.2; no protease present, detectable levelsof Stat-1 protein). The other extract was from a patient with CFS (i.e.,RNase L ratio>2.0; protease(s) present, no detectable level of Stat-1protein).

Sixty (60) micrograms of PBMC extract from the healthy control was mixedwith 60 micrograms of PBMC extract from a CFS patient and incubated at37 C for 15 minutes in the presence of a) buffer only, b) DMSO (solventfor the protease inhibitors), c) Calpastatine, 50 micromolar finalconcentration, d) Calpain Inhibitor Type I, 200 micromolar finalconcentration, e) Calpain Inhibitor Type II, 200 micromolar finalconcentration, f) Caspase 3 Inhibitor Type II, 5 micromolar finalconcentration, or g) Proteasome Inhibitor (MG 132), 50 micromolar finalconcentration. All protease inhibitors above, as well as DMSO, were fromSigma Chemical Company.

After the 15 minute incubation, 2×SDS-PAGE gel sample dye was added andthe sample heated to 95 C for five minutes to denature the proteins. Thedenatured samples were then subjected to standard SDS-PAGE using a 4percent stacking gel and 10 percent separating gel. Transfer to PVDFmembrane, Western blot with anti-Stat-1 antibody, and color developmentwere performed exactly as described above.

II. Analysis of Results

FIG. 1 represents a densitometric scan of a Western blot detectingStat-1 protein and Stat-1 protein fragments from PBMC extracts from CFSpatients. The value indicated at the top of each lane is the ratio ofRNase L fragments as calculated by [(LMW/HMW)*10] as assayed in PBMCextracts from CFS patients. The results demonstrate that the presenceand amount of Stat-1 protein fragmentation directly correlates with thepresence and amount of low molecular weight RNase L fragments in PBMCsamples. These data indicate that native Stat-1 protein is fragmented atan earlier point in the disease cycle than RNase L, and that by the timenative RNase L is demonstrably attacked by proteases (ratio >2.0), thatnative Stat-1 protein has entirely disappeared due to proteolysis,leaving the cells unable to respond to interferons and/or interferoninducers.

FIG. 2 represents a densitometric scan of a Western blot detectingStat-1 protein from PBMC extracts from healthy controls and MS patients.The value indicated at the top of each lane is the ratio of RNase Lfragments as calculated by [(LMW/HMW)*10] as assayed in PBMC extractsfrom CFS patients. The results demonstrate that intact, native Stat-1protein can still be detected in PBMC cell extracts from healthycontrols but not in MS patients, even those with low RNase L ratios(e.g., 0.1, 0.2).

FIG. 3 represents a densitometric scan of a Western blot detecting Bcl-2protein from PBMC extracts from healthy controls and MS patients. As maybe observed, in both healthy controls and MS patients, only minordifferences may be detected in the level of Bcl-2 protein irrespectiveof the RNase L ratio, indicating that the disappearance of Stat-1protein is specific and not an artifact relating to general,non-specific proteolytic degradation.

FIG. 4 represents a densitometric scan of a Western blot detectingStat-1 protein from PBMC extracts when mixed in the presence of variousprotease inhibitors. As may be observed, Stat-1 protein is degraded whena cell extract from a healthy control (i.e., ‘negative extract’; RNase Lratio<0.2, Stat-1 protein-containing) is incubated with a cell extractfrom a patient (RNase L ratio=3.0; Stat-1 protein-negative). Thisdegradation is inhibited in the presence of proteasome inhibitor (MG132) but not in the presence of the other protease inhibitors tested.Thus the degradation of Stat-1 protein is a specific cellular processthat involves the proteasome and does not involve the apoptotic enzymescaspase 3 or calpain.

It is evident from the above results and discussion that relativelysimple and rapid methods for diagnosing and/or characterizing chronicimmune disease (e.g. MS or CFS) activity in a subject are provided bythe subject invention. With the subject methods, accurate diagnosis ofthe chronic immune disease condition, as well the identification of thestage and/or progression of the chronic immune disease condition, may beobtained. As such, the subject methods provide for more accuratediagnostic and/or treatment regimens. In addition, methods of predictingthe effectiveness of particular chronic immune disease treatmentprotocols, e.g., interferon based treatment regiments, are provided,which results in significant improvements in patient care and resourceuse. In addition, methods of treating hosts for chronic immune diseaseare provided. Accordingly, the subject invention represents asignificant contribution to the art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A method for diagnosing whether a host suffers from a chronic immunedisease, said method comprising: (a) assaying a sample from said hostfor the presence of at least one low molecular weight Stat-1 fragment toobtain assay results; and (b) determining whether said host suffers froma chronic immune disease using said assay results; whereby said host isdiagnosed for said chronic immune disease.
 2. The method according toclaim 1, wherein said chronic immune disease is selected from the groupconsisting of CFS and MS.
 3. The method according to claim 1, whereinsaid sample is a blood cell derived sample.
 4. The method according toclaim 1, wherein said sample is a PBMC derived sample.
 5. The methodaccording to claim 1, wherein said host is a human.
 6. A method ofcharacterizing chronic immune disease activity in a human subject, saidmethod comprising: (a) obtaining a sample from said subject; (b)determining the relative amounts of native Stat-1 protein to one or morelow molecular weight Stat-1 protein fragments in said sample; and (c)using said relative amounts to characterize the chronic immune diseaseactivity in said subject.
 7. The method according to claim 6, whereinsaid chronic immune disease is selected from the group consisting of CFSand MS.
 8. The method according to claim 6, wherein said low molecularweight Stat-1 fragment(s) has a molecular weight of approximately 50 kDaunder SDS-PAGE reducing conditions.
 9. The method according to claim 6,wherein said sample is a blood derived sample.
 10. The method accordingto claim 6, wherein said blood derived sample is derived from PBMCs. 11.The method according to claim 6, wherein said method is a method ofconfirming whether said subject suffers from said chronic immunedisease.
 12. A method of characterizing a chronic immune diseaseactivity in a human subject, said method comprising: (a) obtaining asample from said subject; (b) identifying a pattern of low molecularweight Stat-1 fragments in said sample; and (c) using said pattern tocharacterize said chronic immune disease activity in said subject. 13.The method according to claim 12, wherein said chronic immune disease isselected from the group consisting of CFS and MS.
 14. The methodaccording to claim 12, wherein said sample is a blood derived sample.15. The method according to claim 12, wherein said blood derived sampleis derived from PBMCs. 16-39. (canceled)
 40. A method of predicting theeffectiveness of a treatment protocol for a host suffering from achronic immune disease, said method comprising: (a) obtaining a samplefrom said subject; (b) assaying said sample for the presence offull-length Stat-1; and (c) predicting the effectiveness of a treatmentprotocol based upon said assay result.
 41. The method according to claim40, wherein said treatment protocol is an Interferon (IFN)-basedtherapy.
 42. The method according to claim 41, wherein the presence offull-length indicates that said IFN-based therapy will be effective.